1. Field
The present invention relates to a fluorogenic homogeneous assay procedure for determination of high molecular weight molecules in solution.
2. State of the Art
A fluorescent molecule is one which, in response to absorption of light in a characteristic frequency range, emits a photon of longer wavelength. When polarized light is used to stimulate the fluorescence, the light emitted by a plurality of such excited fluorescent molecules is often at least partially polarized, since the emitted photons are emitted at a 90xc2x0 angle with respect to the incident exciting light. This phenomenon is generally termed fluorescence polarization, and can be exploited for quantitation of free vs. analyte-bound fluorescent molecules.
The extent of polarization is a function of several factors including temperature, the rotational mobility of the molecules and solution viscosity. In general, small molecules that rotate rapidly emit light which is less polarized because the emitted light is partially depolarized by the rapid rotation. Very large molecules, however, have limited rotation, which results in a greater degree of polarization.
Fluorescent polarization immunoassays (FPIA or FPI assays) typically use a fluorescently-labelled analyte in conjunction with an antibody which binds the analyte. When the labeled analyte binds to the antibody, the polarization of the fluorescent label increases due to the large hydrodynamic volume of the antigen-antibody conjugate. When unlabelled analyte in a test solution is added to a solution containing the labelled antigen-antibody complex, it competes with the labelled antigen for binding to the antibody. As a result, some of the labelled analyte is displaced into solution, thus decreasing the polarization of the emitted fluorescence. The decrease in polarization occurring upon displacement of labeled analyte is proportional to the amount of unlabeled analyte. The final polarization value may then be used to determine analyte concentration by using a standard curve relating polarization and concentration of unlabelled analyte.
This form of FPI assay has been widely used to determine the concentration of low molecular weight analytes such as drugs and haptens in blood and urine. High molecular weight analytes, however, such as immunoglobulin G (IgG) and human chorionic gonadotrophin (hCG) have been difficult to quantitate with FPIA because the fluorescence polarization value of the free analyte does not differ appreciably from that of the analyte-antibody conjugate. Consequently, the change in polarization as the fluorescently-labelled analyte is displaced by the unlabeled analyte is not sufficient for accurate quantitation.
At least two FPIA methods for measuring high molecular weight antigens exist in the art. U.S. Pat. No. 4,681,859 (Kramer) teaches a method in which a fluorescently labeled small protein or polypeptide is produced that simulates the binding site of a large molecular weight analyte and is capable of binding to the antibody. Because the simulating protein is of relatively low molecular weight, a detectable change in polarization is observed upon its displacement from the antibody by the high molecular weight analyte. The simulator protein of Kramer has the same amino acid sequence as the binding site of the native analyte which binds to the antibody.
Although the method of Kramer may be used to determine the presence of large molecular weight antigens, the accuracy of a quantitation involving the degree of depolarization is more problematic. Factors such as differences in the binding constants between the simulator protein and the native analyte, and the conformation and orientation of the fluorescing compound when the simulator is bound to the antibody, may affect the magnitude of the changes in polarization. Accordingly, the method of Kramer may be improved upon with respect to a quantitative procedure.
U.S. Pat. No. 5,070,025 (Klein et al.) teaches an FPIA process using a fluorescently labeled oligopeptide (xe2x80x9ctracerxe2x80x9d) of 6 to 14 amino acids which is capable of binding to an analyte-specific antibody. The oligopeptide is required to have exactly two cysteine residues which form an intramolecular disulfide bridge. The use of an oligopeptide with a disulfide bridge is typical of early approaches to obtaining an oligopeptide which binds to an antibody with high affinity, in which stabilization of the tertiary configuration of the oligopeptide was deemed necessary. Klein teaches that an otherwise suitable oligopeptide should be altered by appropriate substitutions of amino acids to achieve the result of an oligopeptide which forms a single disulfide bridge in a defined position. Such substitution and the secondary process of causing the bridge to form, is tedious and may affect the fluorescence enhancement and polarization of the labelled oligopeptide when it binds to the antibody. Thus, it is desirable to have an oligopeptide for an FPI assay which does not require stabilization by a disulfide bridge.
Klein et al. further teach that an oligopeptide for an FPI assay should have a molar binding affinity for the antibody which is within a factor of 6 of the binding affinity of the analyte for the antibody. It is often difficult to find oligopeptides whose binding affinity for the antibody is so high compared to the binding affinity of the analyte itself.
Furthermore, the process of Klein is taught to be capable of determining concentrations of insulin on the order of micrograms/milliliter (e.g., micromolar concentrations). However, many analytes are present at concentrations much lower than micrograms per ml, and it would be desirable to be able to detect concentrations as low as nanomolar.
Accordingly, there is a need for improved compositions and methods for fluorescence polarization immunoassay for high molecular weight analytes that provides improved sensitivity and accuracy in quantification. Desirably, an oligopeptide of the composition should be useful even with a binding affinity for the antibody which as much as 100 fold lower than that of the analyte. The method should be rapid, simple and inexpensive to perform, and should be suitable for use in a clinical setting.
The invention is an improved oligopeptide composition for use in a fluorescent polarization immunoassay for a high molecular weight analyte. The composition comprises a fluorescent label bound to an oligopeptide having an amino acid sequence which does not form internal disulfide bridges, a molecular weight which is less than about {fraction (1/500)} of the molecular weight of the antibody which binds the analyte in the immunoassay. In a highly preferred embodiment, the composition exhibits an increase in fluorescence upon binding to the antibody. In another embodiment, the oligopeptide has a binding affinity for the antibody of between about {fraction (1/10)} and {fraction (1/10000)} of the binding affinity of the analyte for the antibody. In still another embodiment, the oligopeptide is selected by a screening procedure in which a plurality of different oligopeptides having respective amino acid sequences that represent sequential overlapping segments of the analyte amino acid sequence. Such a preferred oligopeptide will generally have no more than one cysteine residue.
The invention further embraces a kit and a method for performing a fluorescence polarization assay. The kit includes a monoclonal antibody to an analyte, and an oligopeptide which competes with the analyte for binding to the monoclonal antibody and having an amino acid sequence which is configured to be incapable of formation of an internal disulfide bridge. In a highly preferred embodiment, the oligopeptide is tagged with a fluorophore and the labelled oligopeptide exhibits an increase in fluorescence upon binding to the antibody. In one embodiment, the oligopeptide has a binding affinity for the monoclonal antibody of between about {fraction (1/100)}and {fraction (1/1000)} of the binding affinity of the analyte for the monoclonal antibody.
The kit may further include a buffer suitable for making a solution containing the monoclonal antibody with the oligopeptide. In a further embodiment, the kit is packaged with instructions directing a user to prepare an assay solution containing the monoclonal antibody in a concentration of between about 1xc3x9710xe2x88x928 M and 1xc3x9710xe2x88x927 M and the oligopeptide in a concentration of between about 1xc3x9710xe2x88x929 M and about 1xc3x9710xe2x88x928 M.
In an alternate embodiment, the kit provides the monoclonal antibody and the labelled oligopeptide in an assay solution, with the ratio of oligopeptide to antibody being between about 1:1 and about 1:10. In a further embodiment, the assay solution contains the monoclonal antibody in a concentration of between about 1xc3x9710xe2x88x927 M and 1xc3x9710xe2x88x9210 M and the oligopeptide is present in a concentration of between about 1xc3x9710xe2x88x927 M and about 1xc3x9710xe2x88x9210 M. In another preferred embodiment, the monoclonal antibody concentration is between about 10xe2x88x927 molar and 10xe2x88x928 molar, while the oligopeptide concentration is between about 10xe2x88x928 M and about 10xe2x88x929 M.
A process for a fluorescence polarization immunoassay comprises the steps of: providing a monoclonal antibody which selectively binds an analyte; providing an oligopeptide constructed to selectively bind to the monoclonal antibody, and having an attached fluorescent molecule, the oligopeptide being one of the embodiments described herein; an internal disulfide bridge; contacting the monoclonal antibody with the oligopeptide in solution and determining a first polarization value of the fluorescent molecule; providing a sample comprising an unknown quantity of the analyte; adding the sample to the solution containing the monoclonal antibody and the oligopeptide and determining a second polarization value of the fluorescent molecule; and comparing the first and second polarization values to make an estimate of the amount of the analyte in the sample.
In a further embodiment, in the step of contacting the monoclonal antibody and the oligopeptide the ratio of oligopeptide to antibody is between about 1:1 and about 1:10. In a highly preferred embodiment, quantitation of analyte in the sample is achievable for samples having concentrations of analyte between about 1xc3x9710xe2x88x927 molar and about 1xc3x9710xe2x88x9210 molar.
A screening procedure for the oligopeptide composition includes a test for antibody binding affinity, with the oligopeptide having the highest binding affinity for a monoclonal antibody selectively reactive with the chosen analyte being preferred. The screening procedure may additionally include comparison of the content of proline residues, comparison of the degree of enhancement of fluorescence, and comparison of fluorescence polarization of bound fluorescently-labelled oligopeptide. Generally, each oligopeptide in the series comprises six to ten amino acid residues. One or more oligopeptides having relatively high binding affinities toward the antibody are selected for labelling with a fluorescent molecule. Measurements are made to determine which of the oligopeptide exhibit enhancement of fluorescence of the coupled fluorophore on binding of the oligopeptide to the antibody. Desirably, the oligopeptide is selected both to have a high binding affinity for the antibody, and to provide significant enhancement of fluorescence.
Preferably, the fluorescent label is a dye whose fluorescence occurs at a wavelength providing easy discrimination from the fluorescence of serum which occurs at about 500-515 nanometers (nm). Also desirably, the label is coupled to a side chain near the carboxyl end of the oligopeptide, for example via a free thiol of a cysteine residue.
The invention is exemplified with an oligopeptide and kit designed for an assay for the analyte human chorionic gonadotrophin, but the teachings are readily generalizable to other high molecular weight analytes.